Pin1 is an enzyme that catalyzes isomerization of phosphoserine/threonine-proline (pSer/Thr-Pro) amides. Pin1 is hypothesized to regulate signal transduction leading to mitosis by a conformational switch, isomerizing specific prolyl amides in cell cycle regulatory proteins. Pin1 has also been shown to regulate the activity of Alzheimer's associated tau protein. The unique peptidyl-prolyl isomerase (PPIase) enzymatic activity of Pin1 in the regulation of biological processes is interesting for fundamental reasons. Pin1 consists of two domains that both bind pSer/Thr-Pro motifs with distinct selectivity's. Specific ligands will be developed for each domain separately to be used as tools to study regulation by Pin1. In the first Specific Aim, we propose the design, parallel synthesis and assay of libraries of conformationally constrained trans- proline mimics targeted to the Pin1 WW domain. Parallel synthesis will use solid phase attachment through the invariant phosphate, and amide bond couplings to produce compounds of sufficient purity for screening. A high-throughput competitive enzyme-linked immunosorbent assay (ELISA) will be developed to assay WW domain binding. The WW domain ligands will also be tested for noncompetitive inhibition in the catalytic assay because we have shown inhibition of catalysis by a peptide binding to the WW domain. In the second Aim, libraries will be derived from compounds targeted to the catalytic domain that we have already synthesized: pSer-c/s-Pro isostere, reduced amide, ketone and alpha-ketoamide motifs. The catalytic domain inhibitors will be screened in a high-throughput enzymatic assay, and the mode of inhibition will be determined for the best inhibitors. In the third Aim, the two types of ligands for Pint will be combined into bivalent inhibitors to study the structural and dynamic interactions between the two domains of Pin1. The fourth Aim concerns development of phosphate prodrugs, masked to improve membrane permeability. We will also develop diflurophosphonates stabilized towards phosphatases. These two modifications will allow these compounds to be used in whole cells and in vivo. The best ligands will be useful in collaborations designed to elucidate the structure, function and dynamic relationships within Pin1 and between Pin1 and its ligands.